Blood coagulation is a process consisting of a complex interaction of various blood components, or factors, which eventually gives rise to a fibrin clot. Generally, the blood components that participate in what has been referred to as the coagulation “cascade” are proenzymes or zymogens, enzymatically inactive proteins that are converted to proteolytic enzymes by the action of an activator, which is itself an activated clotting Factor. Coagulation factors that have undergone such a conversion are generally referred to as “active factors”, and are designated by the addition of a lower case “a” suffix (e.g., Factor VIIa).
Activated Factor X (“Xa”) is required to convert prothrombin to thrombin, which then converts fibrinogen to fibrin as a final stage in forming a fibrin clot. There are two systems, or pathways, that promote the activation of Factor X. The “intrinsic pathway” refers to those reactions that lead to thrombin formation through utilisation of factors present only in plasma. A series of protease-mediated activations ultimately generates Factor IXa, which, in conjunction with Factor VIIIa, cleaves Factor X into Xa. An identical proteolysis is effected by Factor VIIa and its co-Factor, tissue factor, in the “extrinsic pathway” of blood coagulation. Tissue factor is a membrane bound protein and does not normally circulate in plasma. Upon vessel disruption, however, it can complex with Factor VIIa to catalyse Factor X activation or Factor IX activation in the presence of Ca2+ and phospholipid. The relative importance of the two coagulation pathways in haemostasis is still unclear.
Factor IXa (FIXa) is a trypsin-like serine protease that serves a key role in haemostasis by generating, as part of the Xase complex, most of the Factor Xa required to support proper thrombin formation during coagulation (reviewed in Hoffman M. and Monroe D. M., III (2001) A cell-based model of hemostasis. Thromb Haemost 85, 958-965). Congenital deficiency of Factor IXa activity is the cause of the X-linked bleeding disorder haemophilia B affecting approximately 1:100,000 males. These haemophilia patients are currently treated by replacement therapy with either recombinant or plasma-derived coagulation Factor IX.
Factor IX is a vitamin K-dependent coagulation factor with structural similarities to Factor VII, Factor X, and protein C. The circulating zymogen form, which has a plasma half-life of about 18-30 hours, consists of 415 amino acids divided into four distinct domains comprising an N-terminal γ-carboxyglutamic acid rich (Gla) domain, two EGF domains, and a C-terminal trypsin-like serine protease domain. Activation of Factor IX occurs by limited proteolysis at Arg145-Ala146 and Arg180-Val181 releasing a 35-aa fragment, the so-called activation peptide (Schmidt A. E. and Bajaj S. P. (2003) Structure-function relationships in Factor IX and Factor IXa. Trends Cardiovasc Med 13, 39-45). The activation peptide is heavily glycosylated containing two N-linked and up to four O-linked glycans.
Prolongation of circulating half-life of proteins can be achieved by modification of the native structure of the proteins. PEGylation is an established method for prolonging the circulating half-life of proteins. GlycoPEGylation of Factor IX (FIX) results in various PEGylated species such as mono-, di and tri-PEGylated species. Such an arrangement therefore provides the possibility for formation of various mono-PEGylated and di-PEGylated species. Mono-PEGylated forms have been identified to possess a desirable pharmacological profile and have therefore been chosen as the preferred drug candidate. It is thus desirable to isolate the mono-PEGylated forms from a mixture of PEGylated and non-PEGylated species.
In addition to separation of PEGylated species from each other and from nonPEGylated species, the purification process must provide sufficient reduction of reagents used in the reaction. It is required to develop a method that ensures the desired product quality and it will be advantageous to develop a single step of purification that can provide sufficient reduction of process related impurities (such as PEGylating reagents, enzymes, by products from reagents) as well as product related impurities (such as nonPEGylated species).
US 2008/207879 (Baxter Int) describes a purification process for rFIX which comprises loading the product onto an anion exchange column and washing the column with a salt concentration of more than 200 mM and eluting with an elution buffer containing divalent cations.